Endomicroscopic optical coherence tomography for cellular resolution imaging of gastrointestinal tracts

Our ability to detect neoplastic changes in gastrointestinal (GI) tracts is limited by the lack of an endomicroscopic imaging tool that provides cellular‐level structural details of GI mucosa over a large tissue area. In this article, we report a fiber‐optic‐based micro‐optical coherence tomography (μOCT) system and demonstrate its capability to acquire cellular‐level details of GI tissue through circumferential scanning. The system achieves an axial resolution of 2.48 μm in air and a transverse resolution of 4.8 μm with a depth‐of‐focus (DOF) of ~150 μm. To mitigate the issue of limited DOF, we used a rigid sheath to maintain a circular lumen and center the distal‐end optics. The sensitivity is tested to be 98.8 dB with an illumination power of 15.6 mW on the sample. With fresh swine colon tissues imaged ex vivo, detailed structures such as crypt lumens and goblet cells can be clearly resolved, demonstrating that this fiber‐optic μOCT system is capable of visualizing cellular‐level morphological features. We also demonstrate that time‐lapsed frame averaging and imaging speckle reduction are essential for clearly visualizing cellular‐level details. Further development of a clinically viable μOCT endomicroscope is likely to improve the diagnostic outcome of GI cancers.      

In vivo detection of tumor boundary using ultrahigh‐resolution optical coherence angiography and fluorescence imaging

Accurate detection of early tumor margin is of great preclinical and clinical implications for predicting the survival rate of subjects and assessing the response of tumor microenvironment to chemotherapy or radiation therapy. Here, we report a multimodality optical imaging study on in vivo detection of tumor boundary by analyzing neoangiogenesis of tumor microenvironment (microangiography), microcirculatory blood flow (optical Doppler tomography) and tumor proliferation (green fluorescent protein [GFP] fluorescence). Microangiography demonstrates superior sensitivity (77.7 ± 6.4%) and specificity (98.2 ± 1.7%) over other imaging technologies (eg, optical coherence tomography) for tumor margin detection. Additionally, we report longitudinal in vivo imaging of tumor progression and show that the abrupt tumor cell proliferation did not occur until local capillary density and cerebral blood flow reached their peak approximately 2 weeks after tumor implantation. The unique capability of longitudinal multimodality imaging of tumor angiogenesis may provide new insights in tumor biology and in vivo assessment of the treatment effects on anti‐angiogenesis therapy for brain cancer.     

En‐face optical coherence tomography for the detection of cancer in prostatectomy specimens: Quantitative analysis in 20 patients

The increase histopathological evaluation of prostatectomy specimens rises the workload on pathologists. Automated histopathology systems, preferably directly on unstained specimens, would accelerate the pathology workflow. In this study, we investigate the potential of quantitative analysis of optical coherence tomography (OCT) to separate benign from malignant prostate tissue automatically. Twenty fixated prostates were cut, from which 54 slices were scanned by OCT. Quantitative OCT metrics (attenuation coefficient, residue, goodness‐of‐fit) were compared for different tissue types, annotated on the histology slides. To avoid misclassification, the poor‐quality slides, and edges of annotations were excluded. Accurate registration of OCT data with histology was achieved in 31 slices. After removing outliers, 56% of the OCT data was compared with histopathology. The quantitative data could not separate malignant from benign tissue. Logistic regression resulted in malignant detection with a sensitivity of 0.80 and a specificity of 0.34. Quantitative OCT analysis should be improved before clinical use.     

Full‐field swept‐source optical coherence tomography and neural tissue classification for deep brain imaging

Optical coherence tomography can differentiate brain regions with intrinsic contrast and at a micron scale resolution. Such a device can be particularly useful as a real‐time neurosurgical guidance tool. We present, to our knowledge, the first full‐field swept‐source optical coherence tomography system operating near a wavelength of 1310 nm. The proof‐of‐concept system was integrated with an endoscopic probe tip, which is compatible with deep brain stimulation keyhole neurosurgery. Neuroimaging experiments were performed on ex vivo brain tissues and in vivo in rat brains. Using classification algorithms involving texture features and optical attenuation, images were successfully classified into three brain tissue types.     

Reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing

Wavefront shaping can compensate the wavefront distortions in deep tissue focusing, leading to an improved penetration depth. However, when using the backscattered signals as the feedback, unexpected compensation bias may be introduced, resulting in focusing position deviations or even no focus in the illumination focal plane. Here we investigated the reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing by measuring the position deviations between the foci in the illumination focal plane and the epi‐detection plane. The experimental results show that when the penetration depth reaches 150 μm in mouse brain tissue (with scattering coefficient ~22.42 mm^?1) using a 488 nm laser and an objective lens with 0.75 numerical aperture, the center of the real focus will deviate out of one radius range of the Airy disk while the optimized focus in the epi‐detection plane maintained basically at the center. With the penetration depth increases, the peak to background ratio of the focus in the illumination focal plane decreases faster than that in the epi‐detection plane. The results indicate that when the penetration depth reaches 150 μm, feedback based on backscattered signals will make wavefront shaping lose its reliability, which may provide a guidance for applications of non‐invasive precise optogenetics or deep tissue optical stimulation using wavefront shaping methods. A, Intensity distribution in the epi‐detection plane and the illumination focal plane before and after correction, corresponding to brain sections with 250 and 300 μm thickness, respectively. Scale bar is 2 μm. B, Averaged focusing deviations in the epi‐detection plane (optimized) and the illumination focal plane (monitored) after compensation. The unit of the ordinate is one Airy disk diameter. Black dashed line represents one Airy disk radius. Bars represent the SE of each measurement set.      

Improved accuracy of quantitative birefringence imaging by polarization sensitive OCT with simple noise correction and its application to neuroimaging

Polarization-sensitive optical coherence tomography (PS-OCT) enables three-dimensional imaging of biological tissues based on the inherent contrast provided by scattering and polarization properties. In fibrous tissue such as the white matter of the brain, PS-OCT allows quantitative mapping of tissue birefringence. For the popular PS-OCT layout using a single circular input state, birefringence measurements are based on a straight-forward evaluation of phase retardation data. However, the accuracy of these measurements strongly depends on the signal-to-noise ratio (SNR) and is prone to mapping artifacts when the SNR is low. Here we present a simple yet effective approach for improving the accuracy of PS-OCT phase retardation and birefringence measurements. By performing a noise bias correction of the detected OCT signal amplitudes, the impact of the noise floor on retardation measurements can be markedly reduced. We present simulation data to illustrate the influence of the noise bias correction on phase retardation measurements and support our analysis with real-world PS-OCT image data.     

Optical coherence hyperspectral microscopy with a single supercontinuum light source

In the paper, we have developed an optical coherence hyperspectral microscopy with a single supercontinuum light source. The microscopy consists of optical coherence tomography (OCT) and hyperspectral imaging (HSI), which can visualize the structural and functional characteristics of biological tissues. The 500 to 700 nm band is selected for HSI and OCT imaging, where HSI enables imaging of oxygen saturation and hemoglobin (Hb) content, while OCT acquires structural characteristics to assess the morphology of biological tissues. The system performance of the optical coherence hyperspectral microscopy is verified by normal mice ears, and the practical applications of the microscopy is further performed in 4T1 and inflammation Balb/c mice ears in vivo. The experimental results demonstrate that the microscopy has potential to provide complementary information for clinical applications.     

Retinal cross-section motion correction in three-dimensional retinal optical coherence tomography

Motion correction is an important issue in ophthalmic optical coherence tomography (OCT), and can improve the ability of data sets to reflect the physiological structures of tissues and make visualization and subsequent analysis easier. In this study, we present a novel method to correct the cross-sectional motion artifacts in retinal OCT volumes. Motion along the x-direction (fast-scan direction) is corrected through the normalized cross-correlation algorithm, while axial motion compensation is performed using the polynomial fitting method on the inner segment/outer segment (IS/OS) layer segmented by the shortest path faster algorithm (SPFA). The results of volunteers with central serous chorioretinopathy demonstrate that the proposed method effectively corrects motion artifacts in OCT volumes and may have potential application value in the evaluation of ophthalmic diseases such as diabetic retinopathy, glaucoma and age-related macular degeneration.     

Endoscopic optical coherence tomography angiography using a forward imaging piezo scanner probe

A forward imaging endoscope for optical coherence tomography angiography (OCTA) featuring a piezoelectric fiber scanner is presented. Imaging is performed with an optical coherence tomography (OCT) system incorporating an akinetic light source with a center wavelength of 1300 nm, bandwidth of 90 nm and A‐line rate of 173 kHz. The endoscope operates in contact mode to avoid motion artifacts, in particular, beneficial for OCTA measurements, and achieves a transversal resolution of 12 μm in air at a rigid probe size of 4 mm in diameter and 11.3 mm in length. A spiral scan pattern is generated at a scanning frequency of 360 Hz to sample a maximum field of view of 1.3 mm. OCT images of a human finger as well as visualization of microvasculature of the human palm are presented both in two and three dimensions. The combination of morphological tissue contrast with qualitative dynamic blood flow information within this endoscopic imaging approach potentially enables improved early diagnostic capabilities of internal organs for diseases such as bladder cancer.      

Advanced fully integrated radiofrequency/optical-coherence-tomography irrigated catheter for atrial fibrillation ablation

The inability of current catheter ablation procedures to accurately monitor lesion formation limits their safety and efficacy. An advanced fully integrated radiofrequency (RF)/optical coherence tomography (OCT) ablation catheter is developed, which enables real-time monitoring during ablation. An OCT fiber array is especially designed, developed and integrated into an off-the-shelf irrigated RF ablation catheter. In-vitro experimental studies performed on poultry and ovine hearts demonstrate the ability of the integrated RF/OCT system to provide information on the quality and orientation of catheter/wall contact. Experimental results show that adipose tissue can be accurately identified from normal myocardial tissue with 94% accuracy and lesion formation is monitored with an overall accuracy of 93%. The ability to predict pop events is also demonstrated, with an accuracy of 86%.     

Gingivitis resolution followed by optical coherence tomography and fluorescence imaging: A case study

Gingivitis is highly prevalent in adults, and if left untreated, can progress to periodontitis. In this article, we present an interesting case study where the resolution of gingivitis was followed over a period of 10 days using optical coherence tomography (OCT) and light-induced autofluorescence (LIAF). We demonstrate that OCT and its functional angiography can distinctively capture the changes during the resolution of gingivitis; while LIAF can detect red-fluorescent signals associated with mature plaque present at the inflamed site. The acute inflammatory region showed evidence of angiogenesis based on the quantification of vessel density and number; while no angiogenesis was detected within the less inflamed region. Gingival thickness showed a reduction of 140 ± 26 μm on average, measured between the peak gingivitis event and the period wherein the inflammation was resolved. Vessels in the angiogenesis site was found to reduce exponentially. The mildly inflamed site showed a decreasing trend in the vessel size, which however was within the error of the measurement.     

Integrated effects of fractional laser microablation and sonophoresis on skin immersion optical clearing in vivo

This study is aimed to find an approach for effective skin optical clearing in vivo using polyethylene glycol 300 (PEG‐300) as an optical clearing agent in combination with physical enhancers: fractional laser microablation (FLMA) and/or low‐frequency sonophoresis. In this study albino outbred rats were used. Light attenuation coefficient and optical clearing potential (OCP) of these approaches were evaluated in upper (from ~70 to ~200 μm) and middle (from ~200 to ~400 μm) dermis separately using optical coherence tomography. In 30 minutes, OCP of sonophoresis in combination with FLMA and PEG‐300 in the upper dermis was the maximal (2.3 ± 0.4) in comparison with other treatments in this time point. The most effective approach for optical clearing of middle dermis was PEG‐300 and sonophoresis; but the maximal value of OCP (1.6 ± 0.1) was achieved only in 90 minutes.     

Optical fan for single‐cell screening

The single‐cell screening has attracted great attentions in advanced biomedicine and tissue biology, especially for the early disease diagnosis and treatment monitoring. In this work, by using a specific‐designed fiber probe with a flat facet, we propose an “optical fan” strategy to screen K562 cells at the single‐cell level from a populations of RBCs. After the 980‐nm laser beam injected into the fiber probe, the RBCs were blown away but holding target K562 cells in place. Further, multiple leukemic cells can be screened from hundreds of red blood cells, providing an efficient approach for the cell screening. The experimental results were interpreted by the numerical simulation, and the stiffness of optical fan was also discussed.     

Feasibility test of a sapphire cryoprobe with optical monitoring of tissue freezing

This article describes a sapphire cryoprobe as a promising solution to the significant problem of modern cryosurgery that is the monitoring of tissue freezing. This probe consists of a sapphire rod manufactured by the edge-defined film-fed growth technique from Al₂O₃ melt and optical fibers accommodated inside the rod and connected to the source and the detector. The probe's design enables detection of spatially resolved diffuse reflected intensities of tissue optical response, which are used for the estimation of tissue freezing depth. The current type of the 12.5-mm diameter sapphire probe cooled down by the liquid nitrogen assumes a superficial cryoablation. The experimental test made by using a gelatin-intralipid tissue phantom shows the feasibility of such concept, revealing the capabilities of monitoring the freezing depth up to 10 mm by the particular instrumentation realization of the probe. This justifies a potential of sapphire-based instruments aided by optical diagnosis in modern cryosurgery.     

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

Nonlinear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtained data were processed using fast Fourier transform (FFT) analysis, while second‐harmonic generation (SHG) anisotropy and the “ratio parameter” values were also calculated. Such measurements were shown to be able to distinguish collagen structure modifications in different cancer grades tested. The analysis presented herein suggests that PSHG imaging could provide a quantitative evaluation of the tumor state and the distinction of malignant from benign breast tissues. The obtained results also allowed the development of a biophysical model, which can explain the aforementioned differentiations and is in agreement with the simulations relating the SHG anisotropy values with the mechanical tension applied to the collagen during cancer progression. The current approach could be a step forward for the development of new, nondestructive, label free optical diagnostic tools for cancer reducing the need of recalls and unnecessary biopsies, while potentially improving cancer detection rates.     

Automatic lumen segmentation using uniqueness of vascular connected region for intravascular optical coherence tomography

We present an automatic lumen segmentation method using uniqueness of connected region for intravascular optical coherence tomography (IVOCT), which can effectively remove the effect on lumen segmentation caused by blood artifacts. Utilizing the uniqueness of vascular wall on A-lines, we detect the A-lines shared by multiple connected regions, identify connected regions generated by blood artifacts using traversal comparison of connected regions' location, shared ratio and area ratio and then remove all artifacts. We compare these three methods by 216 challenging images with severe blood artifacts selected from clinical 1076 IVOCT images. The metrics of the proposed method are evaluated including Dice index, Jaccard index and accuracy of 94.57%, 90.12%, 98.02%. Compared with automatic lumen segmentation based on the previous morphological feature method and widely used dynamic programming method, the metrics of the proposed method are significantly enhanced, especially in challenging images with severe blood artifacts.     

Detecting mouse squamous cell carcinoma from submicron full-field optical coherence tomography images by deep learning

The standard medical practice for cancer diagnosis requires histopathology, which is an invasive and time-consuming procedure. Optical coherence tomography (OCT) is an alternative that is relatively fast, noninvasive, and able to capture three-dimensional structures of epithelial tissue. Unlike most previous OCT systems, which cannot capture crucial cellular-level information for squamous cell carcinoma (SCC) diagnosis, the full-field OCT (FF-OCT) technology used in this paper is able to produce images at sub-micron resolution and thereby facilitates the development of a deep learning algorithm for SCC detection. Experimental results show that the SCC detection algorithm can achieve a classification accuracy of 80% for mouse skin. Using the sub-micron FF-OCT imaging system, the proposed SCC detection algorithm has the potential for in-vivo applications.     

A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus‐oocyte‐complex (COC)

Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non‐invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell‐safe with no inherent cell toxicity or light‐induced phototoxicity indicated by negative DNA damage staining. Pre‐measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01‐0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.     

Lymph vessels visualization from optical coherence tomography data using depth-resolved attenuation coefficient calculation

Multimodal optical coherent tomography grows popularity with researchers and clinicians over the past decade. One of the modalities is lymphangiography, which allows visualization of the lymphatic vessel networks within optical coherence tomography (OCT) imaging volume. In the present study, it is shown that lymphatic vessel visualization obtained from the depth-resolved attenuation coefficient distributions, corrected for the noise, shows improved contrast and detail in comparison with previously proposed approaches. We also argue that the two most popular approaches for lymphatic vessel visualization, namely simple intensity thresholding and vesselness calculation based on local Hessian matrix eigenvalues, imply different definitions of the lymphatic vessel's appearance in the OCT volume and lead to the different networks.     

Restored interlaced volumetric imaging increases image quality and scanning speed during intravital imaging in living mice

Dynamic intravital imaging is essential for revealing ongoing biological phenomena within living organisms and is influenced primarily by several factors: motion artifacts, optical properties and spatial resolution. Conventional imaging quality within a volume, however, is degraded by involuntary movements and trades off between the imaged volume, imaging speed and quality. To balance such trade‐offs incurred by two‐photon excitation microscopy during intravital imaging, we developed a unique combination of interlaced scanning and a simple image restoration algorithm based on biological signal sparsity and a graph Laplacian matrix. This method increases the scanning speed by a factor of four for a field size of 212 μm × 106 μm × 130 μm, and significantly improves the quality of four‐dimensional dynamic volumetric data by preventing irregular artifacts due to the movement observed with conventional methods. Our data suggest this method is robust enough to be applied to multiple types of soft tissue.